Methods for particle characterization (which generally relates to detection as well as other useful characterizations such as location/position determination, particle counting and cell sorting) often suffer from a low signal-to-noise ratio (SNR), since the signal obtained from the particle (in general: a small object) is typically weak in comparison to the background. This is particularly true in connection with optical methods of particle characterization. The low signal-to-noise ratio is also particularly noteworthy in cases of detection of individual particles such as a cell, an aerosol, a molecule, a subvolume of liquid which differs from the surrounding liquid or emulsion, or a piece of DNA with dyes or tags at selection positions.
With respect to the DNA case, conventional DNA sequencing is accomplished by splitting a DNA strand into small pieces, separating the pieces with electrophoresis and then elaborately reconstructing the DNA sequence. An alternative process has recently been developed. In this alternative process, certain base sequences are tagged with fluorescent dyes. After stretching (or “linearizing”) the molecule, the DNA strand is moved through a microfluidic channel at a constant speed. A special fluorescence reader with a high spatial resolution (approx. 1 μm) is used to record the positions of the fluorescent dyes or tags. As a result, an “optical bar code” of the DNA containing the position of the tags is recorded. Therefore, the DNA sequence may be identified.
Typical distance between the tags along the DNA is several μm. Consequently, the required spatial resolution is one μm or better. Typically, this concept is demonstrated by using a con-focal microscope, which allows for exciting and also detecting the fluorescence within a very small volume (˜1 μm3).
FIG. 1 schematically illustrates a conventional approach for spatially resolved fluorescence excitation. As shown, a system 10 includes a detector 12, a channel 14 and an excitation light 16. A small volume within the channel 14 is excited. Light is collected from the excited volume. DNA strings 20 with tagged portions 22 run through an excitation area 24 of the channel 14. The positions of the tags are calculated using a time dependent detector signal.
This approach has been successfully implemented. However, it requires sophisticated and bulky optics to ensure suitably sized excitation and detection volumes. Moreover, the resultant signal-to-noise ratios are lower than desired.